Genetic Evidence for Phospholipid-Mediated Regulation of the

Rab GDP-Dissociation Inhibitor in Fission Yeast

Yan Ma, Takayoshi Kuno, Ayako Kita*, Toshiya Nabata, Satoshi Uno and Reiko Sugiura*

Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome

Sciences, Kobe University Graduate School of Medicine, Kobe, Hyogo, 650-0017, Japan

*Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki

University, Higashi-Osaka, Osaka, 577-8502, Japan

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Genetics: Published Articles Ahead of Print, published on September 15, 2006 as 10.1534/genetics.106.064709

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Running head: Gdi1 and Spo20 in S. Pombe

Key words: GDI, Rab, Spo20, phospholipid, membrane trafficking

Corresponding author: Reiko Sugiura

Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences,

Kinki University, Kowakae 3-4-1, Higashi-Osaka, Osaka, 577-8502, Japan

e-mail: sugiurar@phar.kindai.ac.jp

TEL: +81-6-6730-5880 ext. 3850

FAX: +81-6-6730-1394

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ABSTRACT

We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3

and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast.

In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1+ gene encoding

Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp.

The Gdi1G267D protein failed to extract Rabs from membrane and Rabs were depleted from the

cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1G267D protein was

mostly found in the membrane fraction, whereas wild-type Gdi1 was found in both the

cytosolic and membrane fractions. Notably, overexpression of spo20+, encoding a

phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not

ypt3-i5 or ryh1-i6. The gdi1-i11 and spo20-KC104 mutation are synthetically lethal, and the

wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The

phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the

gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for

the suppression. Furthermore, knockout of the pct1+ gene encoding a choline phosphate

cytidyltransferase rescued gdi1-i11 mutation. Together, our findings suggest that Spo20

modulates Gdi1 function via regulation of phospholipid metabolism of the membranes.

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Rab family small GTPases (Rabs) exist in all eukaryotic cells and form the largest branch

of the small GTPase superfamily (TAKAI et al. 2001). In mammals, Rabs define a family of

almost 70 proteins that play critical roles in the trafficking of vesicles that mediate transport

between compartments of the exocytic and endocytic pathways (PFEFFER 2001; PFEFFER

2005). Like Ras, Rabs act as molecular switches, cycling between an active GTP-bound

state and an inactive GDP-bound state. Thus, transport vesicles bear Rabs with bound GTP;

concomitant with or after membrane fusion, Rabs are converted into their GDP-bound states.

In this manner, target membranes acquire vesicle-derived Rabs in their GDP-bound

conformations (PFEFFER et al. 1995).

We have previously identified two Rab family small GTPases Ypt3/Its5 and Ryh1/Its6 in

fission yeast Schizosaccharomyces pombe (S. pombe) through a genetic screen using the

immunosuppressant drug FK506, a specific inhibitor of calcineurin (CHENG et al. 2002; HE

et al. 2006). In the same genetic screen, we also isolated a mutant of the apm1+ gene that

encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor

protein-1 complex and that is implicated in the Golgi/endosome function (KITA et al. 2004).

This prompted us to further screen immunosuppressant-sensitive mutants to identify

molecules involved in membrane trafficking.

Here, we report the identification and characterization of its11-1/gdi1-i11, a new addition

to the series of immunosuppressant- and temperature-sensitive mutants. The gdi1+ gene

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encodes a protein that is highly similar to the mammalian Rab GDP-dissociation inhibitor

(GDI) (SASAKI et al. 1990) and to Saccharomyces cerevisiae (S. cerevisiae) GDI1/SEC19

(GARRETT et al. 1994). GDI is a Rab-specific regulator that plays a key role in the

recycling of Rabs (ULLRICH et al. 1993). GDI binds and releases Rabs in the presence of

GDP but not GTP, which is thought to be a key biochemical function of GDI to retrieve Rabs

from target membranes. GDI can retrieve a broad spectrum of Rabs from the membrane

following bilayer fusion. In the cytosol, GDI binds Rab in the GDP-bound form. The

heterodimeric complex of GDI and Rab-GDP serves as a cytosolic reservoir to deliver Rab to

the newly formed vesicles, where it becomes activated to the GTP-bound form (LUAN et al.

1999). Thus, GDI plays a key role in the Rab recycling by retrieving prenylated Rabs from

their fusion targets in membranes, maintaining Rabs as a soluble cytosolic complex, and

delivering Rabs to specific membrane-bound compartments for vesicle formation. In

mammals, the importance of GDI function is reflected in the finding that mutations in the

gene encoding the human GDI result in X-linked non-specific mental retardation (D'ADAMO

et al. 1998), and Gdi1-deficient mice show impairment in associative memory and social

behavior (D'ADAMO et al. 2002). In S. cerevisiae, GDI1 is an essential gene, and depletion

of Gdi1p in vivo leads to loss of the soluble pool of Sec4p and inhibition of protein transport

at multiple stages of the secretory pathway (GARRETT et al. 1994).

The structural and mutational analysis of GDI has been focused on the regions called

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sequence conserved regions (SCRs). SCR1 and SCR3B, forming a compact structural unit

at the apex of GDI, were reported to be implicated for Rab binding in vitro and in vivo

(SCHALK et al. 1996; LUAN et al. 1999). In addition to Rab binding, Domain II which

consists of part of SCR2 and SCR3A, is required for both Rab extraction and Rab loading

(GILBERT and BURD 2001). In a study by Luan et al., the double mutation in SCR3B and

SCR3A (R248A-R226A or R248A-Y227A) caused a defect in the association with membrane

(LUAN et al. 2000).

In the present study in fission yeast, we identified a novel mutant allele of the gdi1+ gene

(gdi1-i11), containing an amino acid change at codon 267 (Gly to Asp), which is located

outside the SCRs. The Gdi1G267D protein failed to extract Rabs from the membrane, yet

maintained the ability to bind Rabs. To further delineate Gdi1 function, we screened for

dosage-dependent suppressors of the gdi1-i11 mutant and isolated the spo20+ gene. spo20+ is

an essential gene, which encodes a phosphatidylcholine/phosphatidylinositol transfer protein

(PITP) of the Sec14 family (NAKASE et al. 2001). Further study indicated that the gdi1-i11

and spo20-KC104 mutations are synthetically lethal. Consistently, the wild-type Gdi1 failed

to extract Rabs from the membrane in the spo20-KC104 mutant, indicating that Spo20 is

necessary for Gdi1 to efficiently extract Rabs. We also provide evidence suggesting that the

phosphatidylcholine-transfer activity, but not the phosphatidylinositol-transfer activity, is the

mechanism of suppression of the gdi1-i11 mutation by Spo20. To our knowledge, this paper

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provides the first evidence suggesting that PITP modulates Gdi1 function via regulation of

lipid metabolism.

MATERIALS AND METHODS

Strains, Media, and Genetic and Molecular Biology Methods: The complete

medium YPD and the minimal medium EMM have been described previously (TODA et al.

1996). Standard genetic and recombinant-DNA methods (MORENO et al. 1991) were used

except where noted. FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan).

Isolation of its11-1/gdi1-i11 Mutant and Cloning of the gdi1+ Gene: The

its11-1/gdi1-i11 mutant was isolated in a screen of cells that had been mutagenized with

nitrosoguanidine as described previously (ZHANG et al. 2000). To clone its11+, the its11-1

mutant (KP1892) was grown at 27oC and transformed with an S. pombe genomic DNA library

constructed in the vector pDB248 (BEACH et al. 1982). Leu+ transformants were

replica-plated onto YPD plates at 36oC and the plasmid DNA was recovered from

transformants that showed plasmid-dependent rescue. These plasmids complemented both

the immunosuppressant sensitivity and temperature sensitivity of the its11-1 mutant. By

DNA sequencing, the suppressing plasmids were identified to contain the gdi1+ gene

(SPAC22H10.12c).

To investigate the relationship between the cloned gdi1+ gene and its11-1 mutant,

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linkage analysis was performed as follows. The entire gdi1+ gene was subcloned into the

pUC-derived plasmid containing S. cerevisiae LEU2 gene and integrated by homologous

recombination into the genome of the wild-type strain HM123. The integrant was mated

with the its11-1 mutant. The resulting diploid was sporulated, and tetrads were dissected. A

total of 30 tetrads were dissected. In all cases, only parental ditype tetrads were found,

indicating allelism between the gdi1+ gene and the its11-1 mutation (data not shown).

Cloning of the spo20+ Gene: To screen for dosage-dependent suppressors of its11-1,

the its11-1 mutant (KP1892) was transformed with an S. pombe genomic DNA library, and

Leu+ transformants were replica-plated onto YPD plates at 30oC. By Southern blot analysis,

the suppressing plasmids fell into two classes, with one class containing the gdi1+ gene, and

the other class containing the spo20+ gene (SPBC3H8.10). No other gene was isolated in

this screening.

Tagging of the gdi1+ Gene: The gdi1+ gene was amplified by PCR with genomic DNA

of wild-type cells as a template. The sense primer was 5’-CGC GGA TCC ATG GAT GAG

GAA TAT GAT GTT ATA GTC C-3’, and the antisense primer was 5’-CGC GGA TCC TTA

TTG ATC TTC CAT TTT GGG-3’. The amplified product containing the gdi1+ gene was

digested with BamHI, and the resulting fragment was subcloned into BlueScriptSK (+)

(Stratagene).

For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter

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(MAUNDRELL 1993). Expression was repressed by the addition of 4 µM thiamine to

EMM. To express GST-Gdi1, Gdi1 was tagged at its N-terminus with GST.

GST-Gdi1G267D was made in the same way except that the genomic DNA was from gdi1-i11

mutant cells. Genes either tagged or non-tagged were subcloned into the pREP1 vector to

express the gene (MAUNDRELL 1993).

Gene Deletion: A one-step gene disruption by homologous recombination was

performed (ROTHSTEIN 1983). The gdi1::ura4+ disruption was constructed as follows.

The BamHI fragment containing the gdi1+ gene was subcloned into the BamHI site of

BlueScriptSK (+). Then, a BamHI fragment containing the ura4+ gene was inserted into the

BglII site of the previous construct. The fragment containing the disrupted gdi1+ gene was

transformed into diploid cells. Stable integrants were selected on medium lacking uracil,

and disruption of the gene was checked by genomic Southern hybridization and tetrad

analysis. The pct1+ gene (SPCC1827.02c) was disrupted by the insertion of ura4+ gene at

the HincII site, using a one-step gene disruption by homologous recombination as described

above.

Microscopy: Methods in light microscopy, such as fluorescence microscopy and

differential interference contrast microscopy, were performed as described (KITA et al. 2004).

FM4-64 labeling and staining of vacuoles with Lucifer Yellow (LY) were performed as

described (KITA et al. 2004). Septa were visualized by adding 1 µl of 1 mg/ml Calcofluor

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(Sigma) to a 10-20 µl aliquot of cell suspension. Conventional electron microscopy was

performed as described (KITA et al. 2004).

Assays and Miscellaneous Methods: Tetrad analysis (ZHANG et al. 2000),

expression and detection of GFP-Pho1 (CHENG et al. 2002), cell extract preparation and

immunoblot analysis (SIO et al. 2005) were performed as previously described.

Quantification of the immunoblot data was performed by ImageJ software

(http://rsb.info.nih.gov/ij/). Antibody to fission yeast Cdc4 protein was prepared by

immunizing the rabbit with purified Cdc4 protein, and was used for the detection of

endogenous Cdc4 as a loading control.

RESULTS

Isolation of the its11-1 Mutant: To identify proteins that function in membrane

trafficking, we searched for mutants that are sensitive to the immunosuppressive drug FK506

and isolated the its11-1 mutant (for immunosuppressant- and temperature-sensitive). As

shown in Fig. 1A, its11-1 mutants grew equally well as compared with the wild-type cells at

27oC. However, its11-1 mutant cells could not grow at 36oC nor could they grow on YPD

containing FK506 at the permissive temperature, whereas wild-type cells grew normally (Fig.

1A). As predicted, no double mutant was obtained at any temperature by the genetic cross

between its11-1 and calcineurin deletion (∆ppb1), indicating that its11-1 and ∆ppb1 are

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synthetically lethal (data not shown).

The its11-1/gdi1-i11 is an Allele of the gdi1+ Gene that Encodes a Homolog of

Mammalian GDP-Dissociation Inhibitor: The its11+ gene was cloned by

complementation of the temperature-sensitive growth defect of the its11-1 mutant (Fig. 1A,

YPD36oC, +gdi1+). The its11+ gene also complemented the immunosuppressant sensitivity

of the its11-1 mutant (Fig. 1A, YPD27oC+FK506, +gdi1+). Nucleotide sequencing of the

cloned DNA fragment revealed that its11+ is identical to the gdi1+ gene (SPAC22H10.12c),

which encodes a protein of 440 amino acids that is highly similar to human α-GDI (249/440,

56.59% identity) and S. cerevisiae Gdi1p (272/440, 61.82% identity) (Fig. 1B). Linkage

analysis was performed (see MATERIALS AND METHODS) and results indicated the

allelism between the gdi1+ gene and the its11-1 mutation. We therefore renamed its11-1 as

gdi1-i11. The its11-1 mutant is the only allele of gdi1+ that was identified in the screen of

mutants that show sensitivity to the immunosuppressant.

To identify the mutation in the gdi1-i11 allele, genomic DNA from the gdi1-i11 mutant

was isolated, and the full-length coding region of the gdi1-i11 gene was sequenced. The G

to A nucleotide substitution caused a highly conserved glycine to be altered to an aspartic acid

residue at amino acid position 267 (Fig. 1B, black arrow). We therefore refer to the protein

product of the gdi1-i11 gene as Gdi1G267D. It should be noted that Gly267Asp is a novel

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mutation located outside the SCRs, known to be required for many aspects of GDI function

(Fig. 1C).

Gdi1 is Essential for Cell Viability: To determine the effect of complete loss of gdi1+

function, the gdi1+ gene was knocked out in a wild-type diploid strain by homologous

recombination using the ura4+ gene as a marker (ROTHSTEIN 1983). Southern blotting of

the genomic DNA of one such transformant confirmed that the wild-type gdi1+ gene had been

replaced by the derivative containing the ura4+ insertion (data not shown). Tetrad analysis

indicated that each ascus consisted of two viable and two inviable spores, and that all viable

spores failed to grow in EMM lacking uracil (data not shown). Microscopic observation of

nonviable meiotic progeny showed that these spores germinated but ceased growth soon

thereafter. Therefore, the gdi1+ gene is essential for vegetative cell growth and viability.

Rabs Failed to Distribute in the Cytosolic Fraction in the gdi1-i11 Mutant Cells:

To determine which activity of Rab GDI is affected in the gdi1-i11 mutant, we examined the

distribution of several Rabs by performing immunoblot analyses of the cytosolic and

membrane fractions obtained from wild-type and the gdi1-i11 mutant cells. In wild-type

cells, Ypt3, Ryh1 and Ypt2 predominantly localized to the membrane fraction and were also

found in the cytosolic fraction (Fig. 2, wt). In gdi1-i11 mutant cells, the distribution of the

three Rabs changed from cytosol to membrane (Fig. 2, gdi1-i11). The distribution of Cdc4

as a loading control remained unchanged (Fig. 2, Cdc4). Thus, the gdi1-i11 mutation affects

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the activity of Gdi1 to extract Rabs from the membrane.

The Gdi1G267D Protein Failed to Extract Rabs from the Membrane, yet Maintained

the Ability to Bind Rabs: Glycine267 of the Gdi1 protein is highly conserved from fission

yeast to human. This prompted us to investigate the functional significance of the Gdi1G267D

mutation. To investigate whether Gdi1G267D protein is affected in the ability to bind Rabs,

we expressed GST, GST-tagged Gdi1 or GST-tagged Gdi1G267D protein in GFP-Ypt3

integrated cells, and performed GST pull-down experiments. Expression of GST-Gdi1 fully

suppressed the phenotypes of the gdi1-i11 mutation, indicating that the fusion protein is

functional (data not shown). As shown in Fig. 3A, GST-Gdi1G267D interacted with GFP-Ypt3

as efficiently as GST-Gdi1. GST-Gdi1G267D also interacted with GFP-Ryh1 (data not shown).

The results suggest that Gdi1G267D protein is not affected in the ability to bind Rabs, consistent

with our finding that Gly267 is located outside SCR1 and SCR3B.

Furthermore, to investigate the ability of the Gdi1G267D protein to extract Rabs from

membranes, we expressed GST-fused wild-type gdi1+ gene, the gdi1G267D gene or the control

GST vector in strains chromosomally expressing nmt1-GFP-Ypt3 or nmt1-GFP-Ryh1, and

performed the fractionation experiments. Fractions from the cells were probed with the

anti-GST antibody to detect Gdi1, and probed with the anti-GFP antibody to detect Ypt3 and

Ryh1. As shown in Fig. 3B and 3D, overexpression of wild-type Gdi1 resulted in a

significant increase in the amount of Ypt3 and Ryh1 detected in the cytosolic fraction (+Gdi1),

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whereas overexpression of the Gdi1G267D protein, failed to alter the distribution of these Rabs

between the cytosolic and membrane fractions (+Gdi1G267D). Notably, the Gdi1G267D protein

was detected in the membrane fraction, while it was almost negligible in the cytosolic fraction

(Fig. 3B and 3D, +Gdi1G267D), whereas wild-type Gdi1 was found in both the cytosolic and

membrane fractions (Fig. 3B and 3D, +Gdi1). To exclude the possibility that the

overexpression of Rabs might affect the distribution of Gdi1, we expressed the wild-type

Gdi1 or Gdi1G267D alone in wild-type cells and examined the distribution. Again, the

Gdi1G267D protein was not detected in the cytosolic fraction in wild-type cells (Fig. 3C).

The gdi1-i11 Mutant Cells are Defective for Secretion: The function and genetic

interactions between two Rabs and Gdi1 as described above prompted us to examine the role

of Gdi1 in the secretory pathway, as these two Rabs are involved in Golgi/endosome

membrane trafficking. For this, we examined the ability of gdi1-i11 mutant cells to secrete

SPL-GFP (GFP fused pho1+ leader peptide), because this fusion protein is secreted through

the same pathway as Pho1 acid phosphatase (CHENG et al. 2002).

As shown in Fig. 4, at the permissive temperature, there was no marked decrease in the

amount of secreted SPL-GFP in the growth medium (gdi1-i11, lane 1). However, when

shifted to 33°C for 4 h, the secretion of SPL-GFP was almost abolished in the gdi1-i11 mutant

(gdi1-i11, lane 2), whereas no remarkable change was seen in wild-type cells (wt, lane 2).

These results clearly demonstrate a defect in the secretory pathway associated with the

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gdi1-i11 mutant. On the other hand, there was no dramatic change of secreted SPL-GFP

when the gdi1-i11 mutant was treated with FK506 for 8 h (gdi1-i11, lane 4). This suggests

that calcineurin is not directly involved in the regulation of the secretory pathway. As

calcineurin plays a key role in maintaining cell wall integrity by regulating the enzymes

involved in cell wall synthesis, impairment of both Gdi1 and calcineurin, the two key players

in cell wall synthesis, might explain the synthetic lethal interaction.

Fluid Phase Uptake of Lucifer Yellow is Impaired in gdi1-i11 Mutant Cells: To

assess the effect of the gdi1-i11 mutation on the endocytic pathway, we used the

membrane-impermeable fluorescence dye Lucifer Yellow (LY) to monitor the fluid-phase

endocytosis (RIEZMAN 1985). Wild-type cells and gdi1-i11 mutant cells were incubated in

LY at permissive temperature. At the indicated periods, aliquots were washed extensively to

remove excess LY and the cells were viewed under Nomarski differential interference contrast

and fluorescence microscope. LY clearly labeled the vacuoles of wild-type cells after

30-min incubation (Fig. 4B, wt, 30 min and 60 min). However, the gdi1-i11 mutant cells

showed only weak staining of the cell wall (Fig. 4B, gdi1-i11, 15 min and 30 min) and small

internal structures (Fig. 4B, gdi1-i11, 60 min), indicating that fluid-phase endocytosis is

significantly impaired in gdi1-i11 mutant cells even at the permissive temperature. It should

be noted that, in ypt3-i5 and ryh1-i6 mutant cells, no defect was detected in endocytosis (data

not shown), indicating that Gdi1 has other Rab partner(s) functioning in the endocytic

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pathway.

Abnormal Findings in gdi1-i11 Mutant Cells as Observed in Electron Micrographs:

As noted, gdi1-i11 mutant cells exhibited various defects in membrane trafficking and this

prompted us to perform electron microscopy analysis. Wild-type cells and gdi1-i11 mutant

cells were grown at the permissive temperature of 27oC, and then each culture was divided

into two portions. One portion was maintained at 27oC, while the remaining portion was

shifted to 33oC for 4 h. Cells were then fixed and examined by electron microscopy. At

27oC, the morphology of gdi1-i11 mutant cells was almost indistinguishable from wild-type

cells, except that fragmented vacuoles were often observed (data not shown). Upon

temperature up-shift to 33oC for 4 h, the appearance of gdi1-i11 mutant cells was clearly

distinct from that of wild-type cells (Fig. 4C). First, the accumulation of putative large

post-Golgi vesicles ranging 100-150 nm in size (intensely stained after permanganate

fixation), were observed around the Golgi structures (Fig. 4D, a). These large vesicles were

often observed in gdi1-i11 mutant cells (>50%), whereas they were rarely observed in the

wild-type cells. Second, the abnormal membranous structure, that may likely be Berkeley

body that represents abnormal Golgi membrane, was also observed (Fig. 4D, b). Third, the

abnormal electron-dense membranous structures that were about 500 nm in diameter were

observed and were closely connected to the Golgi structures (Fig 4D, d). These structures

were negligible in wild-type cells. These abnormal membranous structures related to the

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Golgi structures were observed in gdi1-i11 mutant cells (approximately 3%), whereas they

were seldom observed in the wild-type cells. Fourth, almost all the gdi1-i11 mutant cells

showed fragmented vacuoles (Fig 4D, c), whereas the wild-type cells showed larger vacuoles

(data not shown). As observed in the electron micrographs, it should be noted that these

abnormal findings in gdi1-i11 mutant cells are similar to those we previously reported in

ypt3-i5 mutant cells (CHENG et al. 2002). Thus, these findings are consistent with our data

that Ypt3 accumulated in membrane fraction in gdi1-i11 mutant (Fig. 2), and that Gdi1 plays

an important role in recycling of Ypt3.

Gdi1 Shows Genetic Interactions with the Sec14-like PITP Spo20: To identify

proteins that function together with Gdi1, or that bypass the requirement for Gdi1, we

performed a screen for gene dosage-dependent suppressors that could rescue the growth

defect of the gdi1-i11 mutants at the nonpermissive temperature of 30°C. This led to the

isolation of several clones containing the spo20+ gene. As shown in Fig. 5A, the

overexpression of the spo20+ gene suppressed the growth defect of the gdi1-i11 mutants at

30°C, but not at 33°C. The spo20+ gene fully suppressed the immunosuppressant sensitivity

of the gdi1-i11 mutants.

We also investigated whether the overexpression of the spo20+ gene could suppress the

growth defects of ypt3-i5 and ryh1-i6 mutants. As shown in Fig. 5B, overexpression of the

spo20+ gene failed to suppress the temperature sensitivity and immunosuppressant sensitivity

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of these Rab mutants. Thus, Spo20 overexpression suppressed phenotypes of gdi1-i11

mutants, but not those of ypt3-i5 and ryh1-i6 mutants, indicating that Spo20 specifically

suppressed the growth defects of gdi1-i11 mutants.

In fission yeast, the spo20+ gene encodes Sec14-like PITP (NAKASE et al. 2001) which

thus far contains five members including spo20+. Then, we examined the ability of each of

these gene members to suppress the temperature sensitivity and immunosuppressant

sensitivity of the gdi1-i11 mutants. The results clearly showed that overexpression of Spo20,

but not other Sec14 family members, suppressed the phenotypes of the gdi1-i11 mutants (Fig.

5C), indicating that the suppression of the phenotypes of gdi1-i11 mutant was specifically

caused by the overexpression of Spo20.

To further examine the genetic interaction between Gdi1 and Spo20, we crossed the

gdi1-i11 mutant with spo20-KC104 mutant and performed tetrad analysis (Fig. 5D). The

spo20-KC104 mutant is a missense allele of the essential spo20+ gene, and shows

temperature-sensitive growth defect and is defective in cell separation at the restrictive

temperature (NAKASE et al. 2001). The results showed that no double mutant was obtained,

indicating that gdi1-i11 and spo20-KC104 mutation are synthetically lethal, and therefore

Spo20 activity is essential for the viability of gdi1-i11 mutants. Together, these data are

indicative of a highly specific functional relationship between Gdi1 and Spo20.

Spo20 Suppressed the Defective Membrane Trafficking of the gdi1-i11 Mutant

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Cells: To determine if overexpression of Spo20 could rescue the membrane trafficking

defects of the gdi1-i11 mutants, first, we examined the effect of Spo20 overexpression on the

abnormal localization of GFP-fused Syb1, the synaptobrevin in fission yeast (EDAMATSU

and TOYOSHIMA 2003). As a secretory vesicle SNARE, Syb1 would be expected to cycle

between the cell surface and the endocytic pathway. In gdi1-i11 mutants harboring vector,

GFP-Syb1 failed to localize to the cell ends and accumulated as large dots in the cytosol as

compared with wild-type cells (Fig. 6A). While in gdi1-i11 mutants harboring spo20+,

GFP-Syb1 was visible at the cell ends and the accumulation of the large dots in the cytosol

was greatly improved (Fig. 6A, +spo20+). This result indicated that Spo20 overexpression

suppressed the defective GFP-Syb1 localization of gdi1-i11 mutant.

Second, we examined the effect of Spo20 overexpression on vacuole fusion, as

fragmented vacuoles were observed in the electron micrographs (Fig. 4C, c). FM4-64 is a

vital fluorescent dye that is internalized in living cells and then accumulates at the vacuoles

(VIDA and EMR 1995). When the cells were labeled with FM4-64 for 60 min, tiny

vacuoles were observed in the gdi1-i11 mutants (Fig. 6B, -H2O, +vector), while big vacuoles

were observed in wild-type cells (Fig. 6B, -H2O, +gdi1+). In a study by Bone, hypotonic

stress causes a dramatic fusion of vacuoles in S. pombe (BONE et al. 1998). When cells

were collected, washed, and resuspended in water for 60 min, the wild-type cells had

evidently large vacuoles that resulted from vacuole fusion (Fig. 6B, +H2O, +gdi1+), while

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vacuoles remained small and numerous in gdi1-i11 mutants (Fig. 6B, +H2O, +vector). In

contrast, gdi1-i11 mutants transformed with spo20+ contained large vacuoles Fig. 6B, +H2O,

+spo20+), indicating that overexpression of Spo20 partially but clearly suppressed the

defective vacuole fusion of gdi1-i11 mutants.

Finally, we examined the effect of Spo20 overexpression on defective cytokinesis.

Cells grown to mid-log phase at 27°C in liquid YPD medium were subjected to a temperature

up-shift or to the medium containing FK506 at 27°C. Upon shifting to 33°C for 6 h, the

frequency of septated cells in gdi1-i11 mutant cells significantly increased to 46% (Fig. 6C

and 6D, left panel, +vector), whereas the septation index of wild-type cells remained

unchanged (Fig. 6C and 6D, left panel, +gdi1+). Strikingly, the magnitude of the defect in

gdi1-i11 mutant cells were even more pronounced after the addition of FK506 for 6 h,

because nearly 66% of gdi1-i11 mutant cells were septated (Fig. 6C and 6D, right panel,

+vector) as compared with 34% of wild-type cells which were septated (Fig. 6C and 6D, right

panel, +gdi1+). As shown in Fig. 6C and 6D, Spo20 overexpression partially suppressed the

defect in cytokinesis at the restrictive temperature of 33°C. Notably, Spo20 overexpression

almost completely suppressed the defective cytokinesis caused by FK506 treatment.

Phosphatidylinositol-Transfer Activity is Dispensable for the Suppression of the

Phenotypes of gdi1-i11 Mutants: To provide insight into the mechanism by which Spo20

overexpression suppresses the phenotypes of gdi1-i11 mutants, we examined whether Spo20

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influences the ability of Gdi1 to extract Rab from the membrane. The spo20-KC104 mutant

cells integrated with GFP-Ypt3, harboring control vector or pREP1-GST-Gdi1 were cultured

in EMM containing 4 µM thiamine for 8 hours, and then each culture was divided into two

portions. One portion was maintained at 27oC, while the remaining portion was shifted to

36oC for 4 h. The cells were collected and the fractionation experiment was performed.

Notably, in spo20-KC104 mutant cells, Gdi1 failed to extract Ypt3 from membrane when

shifted to 36oC, although it maintains the ability at the permissive temperature of 27oC (Fig.

7A). In wild-type cells, Gdi1 maintains its ability to extract Ypt3 from membrane even at

36oC (data not shown). Thus, Spo20 is necessary for facilitating Gdi1 to extract Rab from

membrane.

In a study by Philips et al, cytosol from Sec14K66A or Sec14K239A-expressing strains

exhibited a reduced phosphatidylinositol-transfer activity, and Sec14K66AK239A-expressing

strains exhibited no detectable phosphatidylinositol-transfer activity, but normal

phosphatidylcholine-transfer activity (PHILLIPS et al. 1999). To examine whether

phosphatidylinositol-transfer activity is necessary for suppression of the gdi1-i11 mutant, we

created three mutants namely Spo20K61A, Spo20K234A and Spo20K61AK234A, based on an

analogy to budding yeast Sec14. As shown in Fig. 7B, all of three mutant versions of Spo20

suppressed FK506- and temperature- sensitive phenotype of gdi1-i11 mutant, and the

suppression was the same as that of wild-type Spo20. The result suggests that

22

phosphatidylinositol-transfer activity is dispensable for the suppression of the phenotypes of

gdi1-i11 mutants.

Deletion of the pct1+ Gene Encoding a Homolog of Choline Phosphate

Cytidyltransferase Suppressed the Phenotypes of gdi1-i11 Mutants: Sec14p, budding

yeast homolog of Spo20, plays a key role in regulating inositol and choline phospholipid

metabolism (LI et al. 2000; HUIJBREGTS et al. 2000). The phosphatidylcholine- and

phosphatidylinositol-bound forms of Sec14p are proposed to independently regulate inositol

and choline phospholipid metabolism (KEARNS et al. 1998). Phosphatidylcholine-bound

Sec14p is hypothesized to down-regulate flux through the diacylglycerol-utilizing

CDP-choline pathway for phosphatidylcholine biosynthesis via inhibiting the activity of

PCT1 gene encoding choline phosphate cytidyltransferase, a rate-limiting enzyme. On the

assumption that overexpression of Spo20 may suppress the phenotypes of gdi1-i11 mutant by

inhibiting Pct1 activity, we examined whether pct1 deletion can rescue the phenotypes of

gdi1-i11 mutants. Expectedly, the growth defect of the gdi1-i11 mutant was rescued by pct1

deletion (Fig. 7C) to the same extent as that of Spo20 overexpression (Fig. 5A), thus

providing further evidence that Spo20 may affect Gdi1 function via modulating

phosphatidylcholine metabolism.

DISCUSSION

23

Our present study highlights the functional significance of the highly conserved Gly267

located outside the SCRs of Gdi1. More importantly, we discovered a genetic and functional

interaction between Gdi1 and genes encoding proteins involved in phospholipid metabolism.

Highly Conserved Gly267 is Important for Gdi1 Function in vivo: Structural and

mutational analyses of Gdi1 have focused on the SCRs. SCR1 and 3B were implicated for

Rab binding in vitro and in vivo (SCHALK et al. 1996; LUAN et al. 1999). In S. cerevisiae,

two mutants were reported to be defective in extracting Rabs from membrane. One mutant

contained multiple mutations located in Domain II, which consists of part of SCR2 and

SCR3A (GILBERT and BURD 2001), and the other mutant contained mutations in both

SCR3B and SCR3A (LUAN et al. 2000). However, the gdi1-i11 single substitution

Gly267Asp mutation is located outside the SCRs, and caused the movement of Rabs from the

cytosol to the membrane, and the overexpression of Gdi1G267D protein failed to increase the

cytosolic pool of Rabs, thus indicating a defect in extracting Rabs from membranes.

However, the Gdi1G267D protein maintains the ability to bind Rabs.

Remarkably, the cytosolic Gdi1G267D protein dramatically decreased, yet maintaining the

ability to associate with the membrane. Gdi1G267D is the first report that an amino acid

change located outside SCRs affected the intracellular distribution of Gdi1, and this might be

a novel regulatory mechanism of Gdi1 function. It may not be merely the charge of the

aspartic acid residue that altered the intracellular Gdi1 distribution; instead, the mutation may

24

cause a conformational change by introducing a bulkier group than the wild-type glycine.

Functional Link between Gdi1 and Sec14-like Phosphatidylinositol Transfer Protein

Spo20: Here, we identified Spo20 as a dosage-dependent suppressor of the gdi1-i11

mutation. Overexpression of Spo20 also suppressed the defects in membrane trafficking

including abnormal Syb1 localization, defective vacuole fusion and defective cytokinesis.

How does the overexpression of Spo20 suppress the phenotypes of gdi1-i11 mutants?

Spo20, similar to S. cerevisiae Sec14p, regulates the Golgi secretory function in fission yeast

(NAKASE et al. 2001). Thus, it might be that Spo20 suppressed phenotypes of gdi1-i11, by

promoting the membrane trafficking from Golgi. However, Spo20 overexpression

suppressed the growth defects of gdi1-i11 mutants, but not those of ypt3-i5 or ryh1-i6 mutant

that demonstrates defect in Golgi membrane trafficking, indicating that the suppression by

Spo20 is highly specific to gdi1-i11 mutants, and that the suppression by Spo20 is not caused

by its effects in Golgi trafficking.

To address the role of Gdi1 and its regulation by Spo20, we examined if the

phosphatidylinositol-transfer activity of Spo20 is required for suppression of gdi1-i11.

Surprisingly, our results showed that phosphatidylinositol-transfer activity is dispensable for

the suppression of gdi1-i11 phenotypes, suggesting that the mechanisms of suppression

include phosphatidylcholine-transfer activity of Spo20. Consistently, the ability to suppress

gdi1-i11 is unique to Spo20 that was reported to exhibit both phosphatidylinositol- and

25

phosphatidylcholine-transfer activity (NAKASE et al. 2001), and is not shared by other PITP

encoding genes, which were reported to exhibit only phosphatidylinositol-transfer activity in

budding yeast.

To further investigate whether Spo20 modulates Gdi1 function, we examined Rab

membrane extraction by Gdi1 in spo20-KC104 mutant cells. The wild-type Gdi1 was able

to extract Rabs from the membrane in the wild-type background, but failed to extract them in

the spo20-KC104 mutant at the restrictive temperature. Thus, the combination of mutations

in spo20-KC104 and gdi1-i11, by causing severe reduction of Rab extraction from membranes,

would disrupt membrane trafficking mediated by Rabs. This might explain the synthetic

lethality between spo20-KC104 and gdi1-i11. According to the study by Fern P. Finger and

Peter Novick, a synthetic lethality can be interpreted as indicating that both genes function in

the same essential pathway if the mutation causes partial loss-of-function in cases where

single null mutations are lethal (FINGER and NOVICK 2000). As single null mutations in

gdi1+ and spo20+ are lethal, the synthetic lethal interaction between gdi1-i11 and

spo20-KC104 indicates that these genes function in the same essential pathway.

As mentioned above, phosphatidylcholine-transfer activity of Spo20 may be important in

that it modulates the lipid composition of the membrane structure, and this in turn affects the

ability of Gdi1 to extract Rab. Consistent with these findings, deletion of the pct1+ gene

which encodes a homolog of choline phosphate cytidyltransferase, a rate-limiting enzyme in

26

the diacylglycerol- utilizing CDP-choline pathway for phosphatidylcholine biosynthesis,

rescued the gdi1-i11 mutation to the same extent as that of Spo20 overexpression. This

further strengthens our hypothesis that Spo20 exerts its effect on gdi1-i11 mutant through

regulation of phospholipid metabolism.

ACKNOWLEDGEMENT

We thank Dr. Takashi Toda, Dr. Mitsuhiro Yanagida, Dr. Chikashi Shimoda, Dr. Kaoru

Takegawa and the National Bio-Resource Project for providing strains and plasmids, Susie O.

Sio for critical reading of the manuscript, and Fujisawa JAPAN Inc. for gifts of FK506. This

work was supported by 21st Century COE Program, the Asahi Glass Foundation, the Uehara

Memorial Foundation and research grants from the Ministry of Education, Culture, Sports,

Science and Technology of Japan.

27

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33

FIGURE LEGENDS

FIG. 1. Mutation in the its11+/gdi1+ gene causes immunosuppressant- and

temperature-sensitive phenotypes. (A) The immunosuppressant and temperature sensitivities

of the its11-1/gdi1-i11 mutant cells. Cells transformed with the multicopy vector pDB248 or

the vector containing the gdi1+ gene were streaked onto each plate containing YPD, YPD plus

0.5 µg/ml FK506, then incubated for 4 days at 27oC or 3 days at 36oC, respectively. (B)

Alignment of protein sequences of S. pombe Gdi1 with related proteins from human and S.

cerevisiae. Sequence alignment was performed using the Clustal W program. Asterisks

indicate identical amino acids. Arrow indicates the mutation site in the glycine267 of Gdi1,

which when mutated to aspartic acid resulted in immunosuppressant- and

temperature-sensitive function in Gdi1. (C) Cartoon of the sequence conserved regions

(SCRs) and where the gdi1-i11 mutation resides. Star indicates the mutation site.

FIG. 2. Rabs failed to distribute in the cytosolic fraction in the gdi1-i11 mutant cells.

Wild-type cells and gdi1-i11 mutant cells transformed with each of the indicated GFP-Rab

were grown in EMM containing 4 µM thiamine for 12 hours. Cells were lysed in the

absence of detergent and the resulting extracts were centrifuged at 100,000 x g for 70 min to

generate membrane and cytosolic fractions. Equal aliquots were treated with antibodies to

34

GFP. Endogenous Cdc4 was used as a loading control and was immunoblotted using

anti-Cdc4 antibodies. C, cytosolic fraction; M, membrane fraction.

Fig. 3. The characterization of the Gdi1G267D protein. (A) Co-precipitation of Ypt3

with GST-Gdi1G267D. GFP-Ypt3 integrated cells expressing pREP1-GST (control),

pREP1-GST-Gdi1 or pREP1-GST-Gdi1 G267D were grown in EMM medium in the absence of

thiamine for 20 hours. The proteins were extracted in the presence of detergent. GST-Gdi1,

GST-Gdi1 G267D and GST were precipitated by glutathione beads, washed extensively, and

subjected to SDS-PAGE and immunoblotted using anti-GFP or anti-GST antibodies. (B)

Distribution of Ypt3 and Ryh1 between membrane and cytosolic fractions after wild-type

Gdi1 and Gdi1G267D overexpression. GFP-Ypt3 and GFP-Ryh1 integrated cells harboring

pREP1-GST-Gdi1 or pREP1-GST-Gdi1G267D were grown in EMM containing 4 µM thiamine

for 12 hours. The cells were collected and the fractionation experiment was performed as

described in Fig. 2. Equal aliquots were treated with anti-GFP antibodies to detect Ypt3 and

Ryh1, and with anti-GST antibodies to detect Gdi1. C, cytosolic fraction; M, membrane

fraction. (C) Distribution of overexpressed wild-type Gdi1 or Gdi1G267D in cytosolic and

membrane fractions in wild-type cells. Wild-type cells harboring pREP1-GST-Gdi1 or

pREP1-GST-Gdi1G267D were grown in EMM containing 4 µM thiamine for 20 hours. The

cells were collected and the fractionation experiment was performed as described in Fig. 2.

35

(D) Quantifications of the distribution of Rabs and GDIs (co-expressed with GFP-Ypt3)

between cytosolic and membrane fractions. The panels in Fig. 3B were quantitated using

ImageJ software.

FIG. 4. Defects in membrane traffic in the gdi1-i11 mutant cells. (A) Defective

secretion of SPL-GFP to the growth medium in gdi1-i11 mutants. Wild-type cells and

gdi1-i11 mutant cells expressing SPL-GFP were cultured at 27 or 33°C for 4 h or at 27°C with

or without the addition of FK506 for 8 h. Proteins were separated by SDS-PAGE and

analyzed by Western blotting using anti-GFP antibody. Media, the trichloroacetic

acid-precipitate of supernatant of 5 x 106 cells; Cells, cell extract of 1 x 106 cells. (B)

Time-course analysis of LY internalization. Wild-type (wt) and gdi1-i11 mutant cells were

incubated in the medium containing Lucifer yellow (5 mg/ml) and the washed cells were

viewed under a fluorescence microscope (LY) and a differential interference contrast

microscope. Bar, 10 µm. (C) Electron microscopic analysis of gdi1-i11 mutant cells grown

at 33°C for 4 h. (D) The boxed regions a, b and c in (C) are enlarged. (a) Putative

post-Golgi vesicles around Golgi structures. Bar, 1 µm. (b) An abnormal membranous

structure of Berkeley body. Bar, 0.5 µm. (c) Fragmented vacuoles in gdi1-i11 mutant. Bar, 1

µm. (d) Abnormal electron-dense membranous structures closely connected to the Golgi

structure. Bar, 1 µm.

36

FIG. 5. The isolation of spo20+ as a specific dosage-dependent suppressor of the

gdi1-i11 mutant cells. (A) and (B) Overexpression of Spo20 suppressed phenotypes of the

gdi1-i11 mutant, but not those of ypt3-i5 or ryh1-i6 mutant. The indicated cells transformed

with the control vector or spo20+ were spotted onto each plate as indicated. Cells were

spotted in serial 10-fold dilutions starting with OD660 = 0.3 of log-phase cells (5 µl). (C)

Overexpression of Spo20, but not other Sec14 family members, suppressed the phenotypes of

the gdi1-i11 mutant. The gdi1-i11 mutants expressing control vector, SPBC23B6.04,

SPBC3H8.02, SPBC365.01, SPBC589.09 and spo20+ were spotted onto the indicated plates,

and then incubated for 4 days at 27oC or 30oC, respectively. (D) The gdi1-i11 mutant is

synthetically lethal with spo20-KC104 mutant. Tetrad analysis of progeny derived from

crossing KP1893 (h+ his2 leu1-32 gdi1-i11) with KP2678 (h– leu1-32 ura4-D18

spo20-KC104). Colonies grown on YPD at 27 °C were replica-plated onto various plates as

indicated. Colonies that failed to grow on YPD at 33°C and at 36°C were predicted to be

gdi1-i11 (gdi1), and those that grew on YPD at 33°C and grew very slowly at 36°C were

predicted to be spo20-KC104 (spo20). The rest of the colonies that grew at either of the

temperatures were predicted to be wild-type cells (wt). Spores that failed to grow were

predicted to be gdi1-i11spo20-KC104 double mutants (D).

37

FIG. 6. Spo20 suppressed the defective phenotypes associated with gdi1-i11 mutants.

(A) Spo20 partially suppressed the defective localization of GFP-Syb1 in the gdi1-i11 mutant

cells. The gdi1-i11 mutant cells expressing chromosome borne GFP-Syb1, were transformed

with the control vector, gdi1+ or spo20+, and were examined under the fluorescence

microscope. (B) Spo20 suppressed the defective vacuole fusion in the gdi1-i11 mutant cells.

The gdi1-i11 mutant cells transformed with each of the indicated plasmid were grown in YPD

medium at 27°C. Cells were harvested, labeled with FM4-64 fluorescent dye for 60 minutes

(see MATERIALS AND METHODS), and then resuspended in water, and examined by

fluorescence microscopy. Photographs were taken after 60 min. Bar, 10 µm. (C) Spo20

suppressed the defective cytokinesis in the gdi1-i11 mutant cells. The gdi1-i11 mutant cells

expressing control vector, spo20+ or gdi1+ were shifted to the restrictive temperature (33°C)

for 6 h, or FK506 was added for 6 h and incubated at 27°C and then stained with Calcofluor

to visualize cell wall and septum. Bar, 10 µm. (D) Percentage of cells forming a division

septum at each time point in the gdi1-i11 mutant cells expressing control vector, spo20+ or

gdi1+ after the shift to 33°C (left panel) or the addition of FK506 at 27°C (right panel).

Values are the average of three independent experiments with 500 cells counted for each time

point. Standard deviations between experiments were <10%.

FIG. 7. Spo20 modulates Gdi1 function via regulation of phospholipid metabolism.

38

(A) Spo20 affects extraction of Rab from membrane. The spo20-KC104 mutant cells

integrated with GFP-Ypt3 harboring control vector or pREP1-GST-Gdi1 were cultured in

EMM containing 4 µM thiamine for 8 hours, and then each culture was divided into two

portions. One portion was maintained at 27oC, while the remaining portion was shifted to

36oC for 4 h. The cells were collected and the fractionation experiment was performed as

described in Fig. 2. Endogenous Cdc4 was used as a loading control and was

immunoblotted using anti-Cdc4 antiserum. C, cytosolic fraction; M, membrane fraction.

(B) phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of

gdi1-i11 mutant phenotype. The gdi1-i11 mutants expressing the control vector,

Spo20K60AK234A, Spo20K60A, Spo20K234A or wild-type Spo20 were spotted onto the plates as

indicated, and then incubated for 4 days at 27oC or 30oC, respectively. (C) The growth

defect of the gdi1-i11 mutant was partially rescued by pct1 deletion. Wild-type, gdi1-i11

mutant, ∆pct1 and gdi1-i11∆pct1 cells were spotted onto each plate as indicated, and then

incubated for 4 days at 27oC or 30oC, or 3 days at 33oC, respectively.

39

TABLE I

Schizosaccharomyces pombe strains used in this study

Strain Genotype Reference

HM123 h- leu1-32 Our stock

KP162 h- leu1-32 ypt3-i5 Our stock

HM528 h+ his2 Our stock

KP928 h+ his2 leu1-32 ura4-D18 Our stock

KP1248 h- leu1-32 ura4-294 Our stock

KP1259 h + leu1-32 ura4-294 nmt1GFP-ypt3::ura4+ Our stock

KP1892 h- leu1-32 gdi1-i11 This study

KP1893 h+ his2 leu1-32 gdi1-i11 This study

KP2035 h- leu1-32 ura4-294 nmt1GFP-syb1::ura4+ Our stock

KP2439 h- leu1-32 ura4-294 gdi1 nmt1GFP-syb1::ura4+ This study

KP2454 h+ his2 leu1-32 ura4-D18 gdi1-i11 This study

KP2580 h- leu1-32 ura4-D18 ryh1::ura4+ Our stock

KP2591 h-/h+ leu1-32/leu1-32 ura4-D18/ura4-D18 his2/+

ade6-M210/ade6-M216 gdi1+/gdi1::ura4+

This study

KP2678 h– leu1-32 ura4-D18 spo20-KC104 (NAKASE et al. 2001)

KP2881 h + leu1-32 ura4 spo20-KC104

nmt1GFP-ypt3::ura4+

This study

KP2902 h- leu1-32 pct1::ura4+ This study

KP2913 h- leu1-32 ura4-D18 gdi1-i11 pct1::ura4+ This study

A

B

Figure 1 Ma et al.

oYPD 27 C

oYPD 27 C+FK506

oYPD 36 C

its11-1/gdi1-i11

++ gdi1

its11-1/gdi1-i11 + vector

wt+vector

D

CG267DSCR1 SCR2 SCR3

Figure 2 Ma et al.

C M C Mwt gdi1-i11

GFP-Ryh1　

GFP-Ypt3

GFP-Ypt2

Cdc4

Cdc4

Cdc4

Figure 3ABC Ma et al.

C

GFP-Ypt3G267D +GST-Gdi1

C M C M C M

B

wt

+vector +GST-Gdi1

Anti-GFP

Anti-GST

GFP-Ryh1

Anti-GFP

Anti-GST

G267D +GST-Gdi1C M C M C M +vector +GST-Gdi1

A

GST-Gdi1

GFP-Ypt3　

GST

Lysates Beads　　

Expression　　

GST-Gdi1

GFP-Ypt3　

GST

+ + + +- + -

+-+ -G267D

GST-Gdi1 - +- +

+ +

- -- + -

- -

Anti-GST

G267D GST-Gdi1GST-Gdi1

C M C M

Figure 3D Ma et al.

G267D +GST-Gdi1

G267D +GST-Gdi1

CC

C

C

C

C

C

C

M

M

M

M

M

M

MM

+vector

+vector

+GST-Gdi1

+GST-Gdi1

80

80

80

60

60

60

40

40

40

20

20

20

0

0

0

100

100

100

GFP-Ryh1

GFP-Ypt3

G267DGST-Gdi1 GST-Gdi1

DF

racti

ons

of R

abs

in C

yto

sol and

Mem

bra

ne (

%)

Fra

cti

ons

of G

DIs

in

Cyto

sol and

Mem

bra

ne (

%)

F

6 h

K50

8

o

2 C 4

h

7

6

FK50

8hwt

Intra

Extra

A

gdi1-i11

Intra

Extra

o C

4

33

h

o

33

C 4h

o

27

C 8h

o

27 C 8

h

1 2 3 4

1 2 3 4

o

27

C 4h

Figure 4A Ma et al.

wt DIC LY

0min

15min

30min

60min

Figure 4B Ma et al.

gdi1-i11 DIC LY

B

C

Figure 4C Ma et al.

gdi1-i11

a

b

c

Figure 4D Ma et al.

a

d

b

c

D

A

B

YPD + FK506o27 C

YPD + 0.1 M CaCl2o27 C

Figure 5AB Ma et al.

wt+vector

gdi1-i11+vector

+ gdi1-i11+pspo20

wt+vector

gdi1-i11+vector

+ gdi1-i11+pspo20

o 27 C

o 30 C

o 33 C

o 27 C

o 36 C

wt+vector

ryh1-i6+vector+ryh1-i6+pspo20

ypt3-i5+vector+ypt3-i5+pspo20

wt+vector

ryh1-i6+vector+ryh1-i6+pspo20

ypt3-i5+vector+ypt3-i5+pspo20

o + FK506 27 C

o + FK506 27 C

Figure 5CD Ma et al.

D

o YPD 36 C

o YPD 27 C

o YPD 33 C

1 2 3 4 5 6 a b

D

D c d

wt wt

wt

D D wt

D wt

wt

D D wt

gdi1

spo20

gdi1

spo20

gdi1spo20

gdi1

spo20 gdi1

spo20

b

c

d

a

b

c

d

1 2 3 4 5 6 a

2 3 4 5 6 1

Cgdi1-i11 vector+

wt+vector

gdi1-i11+365.01

gdi1-i11+589.09

gdi1-i11+3H8.02

gdi1-i11+23B6.04

+gdi1-i11+spo20o

30 Co

27 C +FK506o

27 C

Figure 6A Ma et al.

A

+ +gdi1

++spo20

+vector

GFP-Syb1 DIC

Figure 6B Ma et al.

B

+ +gdi1

++spo20

+vector

FM4-64+H2O -H2O

Figure 6CD Ma et al.

C

D

6 4 20

30

20

10

Hours after FK506 addition

o Hours at 33 C

Septa

tion

(%)　

40

50

+ +gdi1

+vector++spo20

6 4 20

30

20

10

Septa

tion

(%)　

40

50

60

70

+ +gdi1

+vector++spo20

0 h 6 h

+vector

++spo20

0 h 6 h

o+ FK506 27 C o 33 C

+ +gdi1

Figure 7A Ma et al.

A

GFP-Ypt3

Cdc4

C M C M+control

C M C M

spo20-KC GFP-Ypt30at 27 C

+GST-Gdi1 +control

0at 36 C +GST-Gdi1

Figure 7BC Ma et al.

Bwt + vector

gdi1-i11 + vector

K61AK234A +Spo20

YPD o

at 27 C

K61A +Spo20

+Spo20(wt)

K234A +Spo20

EMM o

at 30 C(OP)EMM+FK506

oat 27 C(OP)

wt

gdi1-i11

gdi1-i11Dpct1

Dpct1

o 27 C o 27 C + FK506

o 30 C

o 33 C

wt

gdi1-i11

gdi1-i11Dpct1

Dpct1

C